Polymerase chain reaction (PCR) is an excellent method, and its field of application increases every year (Randall K. Saiki et al. (1988) Science 239, 487-491). In PCR, it is also possible to amplify DNA fragment starting with just of 1 molecule. A method in which an amplified product of PCR is sequenced without cloning (direct sequencing method) is also useful (Corinne Wong et al. (1988) Nature, 330, 384-386). This method requires neither preparation nor screening of a library, and it is a rapid method enabling to simultaneously obtain sequence information of multiple samples.
Moreover, the inventor introduced a completely novel DNA sequencing method which does not require to remove remaining unreacted primers and 2'deoxyribonucleoside5'triphosphate (2' dNTPs), and which does not require denaturation so that the problem of quick regeneration of PCR products could be obviated [WO96/14434]. This method is a direct sequencing method using RNA polymerase such as T7 RNA polymerase and terminators for RNA transcription reaction (e.g. 3'deoxyribonucleoside5'triphosphate, 3'dNTPs).
The above-mentioned direct transcription sequencing method is performed as described below. RNA polymerase is reacted in a mixture of ribonucleoside5'triphosphates (NTPs) and deoxyribonucleotide(s) (3' dNTP(s)) using DNA amplified by PCR method and the like as a template. In this reaction, ribonucleotides having the bases corresponding to the template DNA sequence are incorporated into a ribonucleotide sequence, termination occurs within corporation of 3'deoxyribonucleotide, and as a result a polynucleotide is synthesized. Resulted polyribonucleotides (nucleic acid transcription products) are separated, and the DNA sequence is determined by analyzing nucleic acid sequence of the separated fraction. Specifically, using florescence labeled 3' dNTP derivatives as a terminator of nucleic acid transcription, nucleic acid sequence can be easily determined by analyzing the label which has been incorporated as a part of the terminator.
By this method, the nucleic acid sequence of PCR-amplified DNA products can be directly used for sequencing without having to remove primers and 2'deoxyribonucleoside5'triphosphates (2' dNTPs). This is because 2' dNTPs do not work as substrates for RNA polymerase. Furthermore, since no denaturation is required, the problem of quick regeneration of the PCR products can be avoided. Therefore the method is extremely powerful.
In the case that a large amount of nucleotide sequence such as the human genome is to be analyzed, a method much more rapid and easier than the existing methods is necessary in order to obtain results in a short time. The above-mentioned direct transcript sequencing method is a relatively rapid method compared to previous sequencing methods utilizing DNA polymerase, however an even more rapid and easier method is necessary. Therefore, it can be thought that a DNA amplification with polymerase chain reaction and a nucleic acid transcript reaction may take place in parallel in the same reaction solution using the above direct transcript sequencing method enabling sequencing rapidly and easily.
However, thepolymerase chain reaction requires an increase or decrease of the temperature of the reaction solution for an amplification of DNA fragments. Therefore, use of thermo-resistant DNA polymerase is required for the polymerase chain reaction. Thus RNA polymerase used for nucleic acid transcript reaction is also required to be thermo-resistant. The above combination method will be possibly performed if a thermo-resistant RNA polymerase having the thermo-resistance similar to that of DNA polymerase would be available. However, at present such thermo-resistant RNA polymerase is not known.
Therefore, an object of the present invention is to provide a method for sequencing DNA in which target DNA amplification and nucleic transcript generation can be operated simultaneously in parallel without use of thermo-resistant RNA polymerase.